The present invention pertains to compositions and methods for the use and synthesis of the compositions. The present invention includes modified nucleotide compositions having linking groups capable of associating with label moieties.
The following definitions are provided to facilitate an understanding of the present invention. The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to a target strand.
The term "linking group" is used broadly to denote a hydrocarbon moiety capable of reacting with a nucleotide or nucleotide derivative and another compound.
The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, and dyes. The term "agent" is used in a broad sense, including any molecular moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any molecular moiety which participates in reactions with the agent.
Genetic information is stored in living cells in threadlike molecules of DNA. In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenosine in an opposing complementary strand.
DNA consists of covalently linked chains of deoxyribonucleotides and RNA consists of covalently linked chains of ribonucleotides. The genetic code of a living organism is carried upon the DNA strand in the sequence of the base pairs.
Each nucleic acid is linked by a phosphodiester bridge between the five prime hydroxyl group of the sugar of one nucleotide and the three prime hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal and having a free five prime hydroxyl group and another terminal end having a three prime hydroxyl group. The terminal ends of polynucleotides are often referred to as being five prime termini or three prime termini in reference to the respective free hydroxyl group. Complementary strands of DNA and RNA form antiparallel complexes in which the three prime terminal end of one strand is oriented and bound to the five prime terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at complementary regions. Presently, nucleic acid hybridization assays are primarily used to detect and identify unique DNA or RNA base sequences or specific genes in a complete DNA molecule, in mixtures of nucleic acid, or in mixtures of nucleic acid fragments.
The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples, may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility, cancer and precancerous states, or bacterial or viral infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacteria, bacterial cultures or tissue containing bacteria may indicate the presence of antibiotic resistance, toxins, viruses, or plasmids, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exist in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxin producing bacteria.
One of the most widely used nucleic acid hybridization assay procedures is known as the Southern blot filter hybridization method or simply, the Southern procedure (Southern, E., J. Mol. Biol. I, 98,503, (1975). The Southern procedure is used to identify target DNA or RNA sequences. This procedure is generally carried out by immobilizing sample RNA or DNA to nitrocellulose sheets. The immobilized sample RNA or DNA is contacted with radio-labeled o--labeled probe strands of DNA having a base sequence complementary to the target sequence carrying a radioactive moiety which can be detected. Hybridization between the probe and the sample DNA is allowed to take place.
The hybridization process is generally very specific. The labeled probe will not combine with sample DNA or RNA if the two nucleotide entities do not share substantial complementary base pair organization. Hybridization can take from three to 48 hours depending on given conditions.
Unhybridized DNA probe is subsequently washed away. The nitrocellulose sheet is placed on a sheet of X-ray film and allowed to expose. The X-ray film is developed with the exposed areas of the film identifying DNA fragments which have been hybridized to the DNA probe and therefore have base pair sequence of interest.
The use of radioactive labeling agents in conjunction with Southern assay techniques have allowed the application of nucleic acid assays to clinical samples. However, the use of radioactive labeling techniques requires a long exposure time to visualize bands on X-ray film. A typical Southern procedure may require 1 to 7 days for exposure. The use of radioactive labeling agents further requires special laboratory procedures and licenses.
The above problems associated with assays involving radioisotopic labels have led to the development of techniques employing nonisotopic labels. Examples of nonisotopic labels include enzymes, luminescent agents, and dyes. Luminescent labels emit light upon exitation by an external energy source and may be grouped into categories dependent upon the source of the exciting energy, including: radioluminescent labels deriving energy from high energy particles; chemiluminescent labels which obtain energy from chemical reactions; bioluminescent labels wherein the exciting energy is applied in a biological system; and photoluminescent or fluorescent labels which are excitable by units of electromagnetic radiation (photons) of infrared, visable, or ultraviolet light. See, generally, Smith et al., Ann Clin. Biochem., 18: 253, 274 (1981).
Nonisotopic assay techniques employing labels excitable by nonradioactive energy sources avoid the health hazards and licensing problems encountered with radio-isotopic label assay techniques. Moreover, nonisotopic assay techniques hold promise for rapid detection avoiding the long exposure time associated with the use of X-ray film.
However, nonradioisotopic assays have not conveyed the sensitivity or specificity required of assays intended for the clinical market. In part, the problem lies in associating a label to a probe without interferring with the hybridization process. A further problem includes the associating of a label to a probe without interferring with the performance of the label.
DNA, unlike proteins, does not contain a variety of organic functional groups which are amenable to direct chemical labeling. An essential pre-requisite therefore to the preparation of non-radioisotopically labeled DNA probes is the chemical manipulation of DNA either at the poly or mononucleotide level such that suitable functionality becomes available for label attachment. Several synthetic methods are described in the literature which enable labeling of DNA with various label groups, for example, Biotinylation: Langer, P. R., Waldrop, A. A. and Ward, D. C. Proc. Nat'l. Acad. Sci. U.S.A. 78(11), 6633, 1981; Kempe, T., Sundquist, W. I., Chow, F. and Hu, S-L., Nucl. Acid. Res. 13(1), 45, 1985; Aliphatic amine group attachment: Chu, B. C. F., Wahl, G. M. and Orgel, L. E., Nucl. Acid. Res. (18), 6513, 1983; Dreyer, G. B. and Dervan P. B., Proc. Nat'l. Acad. Sci. U.S.A., 82, 968, 1985; Sulfhydryl group attachment: Eshaghpour, H., Soll, D. and Crothers, D. M., Nuc'l. Acid. Res., 7(6), 1485, 1979; and Conolly, B. A. and Rider, P., Nucl. Acid. Res. 13 (12), 4485, 1985. However, the synthetic techniques described in the literature involve complex multistage procedures which employ toxic and/or expensive heavy metal reagents.